The present relates generally to the imaging of turbid (i.e., highly scattering) media and more particulary to a novel method and apparatus for the three-dimensional imaging of turbid media, such as biological tissues.
Optical imaging and microscopy have attracted considerable attention because of their potential in the development of non-invasive medical diagnostic modalities. See e.g., Huang et al., Science 254, 1178 (1991); Piston et al., J. Microsc., 178, 20 (1994); Freund et al., Biophys. J., 50, 693 (1986); Benaron et al., Science, 259, 1463 (1993); and Wang et al., Science, 253, 769 (1991), all of which are incorporated herein by reference. Achieving high spatial resolution remains one of the top priorities for precisely localizing biological structures and changes in the state of tissues at different locations. Some of the powerful in vitro and in vivo imaging techniques developed for highly turbid media, to-date, include optical coherence tomography (OCT), time of flight and Fourier-Kerr gate imaging methods, with micrometer to sub-millimeter spatial resolutions. Imaging techniques that use nonlinear-optical effects have been demonstrated to have an additional advantage in spatial resolution, owing to a higher-order dependence on the excitation intensity. Submicrometer lateral resolution has been achieved in three dimensions in the detection of cellular metabolism in the rabbit cornea, through two-photon excitation of fluorescence (TPF) from reduced pyridine nucleotides. A combination of confocal linear-optical approaches and TPF has also been used as an alternative for visualizing the structure of biological tissues. See Denk, J. Biomed. Opt., 1, 296 (1996), which is incorporated herein by reference; see also U.S. Pat. No. 5,034,613, inventors Denk et al., which issued Jul. 23, 1991, and which is incorporated herein by reference.
Second-harmonic generation (SHG) in nearly transparent tissues was first disclosed in Fine et al., Appl. Opt., 10, 2350 (1971), which is incorporated herein by reference. Cross-beam-scanning SHG microscopy was studied with a transmission geometry to show detailed variation of collagenous filaments in a rat tail tendon. Recently, a correlation of second-harmonic signal strength with tissue structure in native chicken tissues was disclosed in Guo et al., "Optical harmonic generation from animal tissues by the use of picosecond and femtosecond laser pulses," Appl. Opt., 35, 6810 (1996), which is incorporated herein by reference. See also Guo et al., "Two photon excitation of fluorescence from chicken tissue," Appl. Opt., 36, 968-970 (1997), which is incorporated herein by reference. In terms of spatial resolution, second-harmonic tomography is identical to two-photon microscopy, in which the localization effect is based on quadratic dependence of the signal on the input photon density. However, an advantage of using an infrared excitation source in second-harmonic tomography is its deeper penetration depth and the fact that it generates less photobleaching and causes less damage than a single-photon-fluorescence confocal microscopy. In contrast with TPF, second-harmonic generation has the advantage that contrast can be obtained from nonfluorescent samples and tissues. An inverse higher-order dependence of second-harmonic intensity on the refractive index allows one to highlight small changes in reflectance. The second-harmonic signal arises from the second-order nonlinear-optical susceptibility .chi..sup.2 tensor, which depends on the electronic configuration, molecular symmetry, local morphology, orientation, and alignment of the molecules and ultrastructures. The potential of using second-harmonic generation to determine symmetry properties of the local environment and surfaces in homogeneous and amorphous media has been demonstrated in Heinz et al., Phys. Rev. Lett., 48, 478 (1982), which is incorporated herein by reference. The excitation wavelength of second-harmonic generation is not restricted to the absorption band of the molecules and thus can be further extended toward the infrared region. This property is in contrast with multiphoton microscopy, in which extending the source wavelength is accompanied by a trade-off in the signal magnitude, through a three-photon or even higher-order process. Second-harmonic generation is a second-order nonlinear-optical process that can generate signals that are orders of magnitude higher than that from a third-order process (TPF), permitting signal detection from deeper in the scattering medium.